The invention relates to a procedure for specific replacement of a copy of a gene present in the genome of a recipient eucaryotic organism by the integration of a gene different from the inactivated gene. Preferably, the recipient gene will be present in at least 2 copies in the transfected host cell. The recipient gene is defined as being the gene where the insertion of the different gene is made.
More particularly, the invention relates to the production of transgenic animals in which the foreign gene has been introduced in a targetted manner in order to make possible both the maintainance of the normal genetic functions of the animal and the expression of the foreign gene under the control of endogenous promoters.
By xe2x80x9cdifferent or foreign genexe2x80x9d is meant any nucleotide sequence corresponding to the totality or a part of a xe2x80x9cforeign or differentxe2x80x9d gene from the recipient gene such as is normally found in the genome (RNA or DNA), or it also corresponds to an artificially modified sequence of the normal gene or also to a fragment of this sequence.
The invention also relates to the process for the production of these transgenic animals.
In the production of transgenic animals, the conventional methods used for the introduction of heterologous DNA sequences into the germinal cell line do not make it possible to control the site of integration of the foreign gene into the genome nor the number of copies thus introduced. The integration of the foreign gene occurs at random and, usually, several copies of the gene are integrated at the same time, sometimes in the form of a head-to-tail tandem, the site of integration and the number of copies integrated varying from one transgenic animal to another.
Thus, it may happen that endogenous cellular genes, situated at the point of insertion, are thus inactivated without this being easily detectable on account of the many random insertions. If the product of these genes is important for the development of the animal, the latter will be seriously perturbed. Moreover, the random insertion of the foreign gene may occur at a site which is not suitable for the expression of the gene. In addition, the fact that there may be variation in the site and in the number of insertions from animal to animal makes the interpretation of the studies of expression extremely difficult.
A major problem encountered in the production of transgenic animals is the obtaining of the expression of the foreign gene. Generally speaking, two types of experiment have been made in mice.
The genes introduced into the germ line are:
either xe2x80x9ccompletexe2x80x9d genes, comprising coding sequences flanked by their own regulatory sequences;
or composite genes, composed of the coding sequence of a gene fused to a promoter sequence of another gene, the two fragments even sometimes belonging to two different animal species.
Thus, it has been possible to confirm that the specificity of the expression of the genes in this or that tissue is determined by their regulatory sequence(s).
The choice of the suitable promoter for the expression of the foreign gene in the transgenic animal is thus of primordial importance.
Furthermore, the directed mutagenesis of mouse genes in embryonic stem cells has recently been carried out by resorting to a technique of xe2x80x9cgene targettingxe2x80x9d (Thomas et al., 1987; Thompson et al., 1989).
In the first case, the mouse HPRT gene was mutated by insertion and replacement and, in the second case, a mutated HPRT gene was corrected. Thompson et al. have extended their experiments to the production of chimeric mice and have observed the passage of the genetic modification in the germ cell line.
In each of the documents cited, the precise site of integration was targetted by homologous recombination between, on the one hand, exogenous sequences bearing the mutation or correction included in a vector under the control of an exogenous promoter and, on the other hand, their genomic homologue. This being so, it should be noted that the earlier authors carried out their experiments on a specific gene (HPRT), the activation of which by mutation is accompanied by a detectable phenotype. The targetted mutation described by Thomas et al. had the effect of inactivating the HPRT gene and, consequently, of causing the normally detectable phenotype associated with the HPRT to disappear. The selection gene NeoR, under the control of a promoter TK, was thus incorporated into the DNA to be inserted in order to make possible the selection of the transformants. It is to be noted that the experiments described in the prior art implied a selection by means of the recipient gene (e.g. HPRT) or by means of the inserted gene (e.g. NeoR). The site of the insertion and/or the type of gene inserted is thus limited to genes conferring a selectable character.
Furthermore, in the prior art, the exogenous sequences on the vector thus serve both to target the integration site and to introduce the modification. Subsequent to homologous recombination, the modified gene is always found in its normal genetic environment.
Let it be recalled that a problem which arises in the course of the production of transgenic animals is the danger of inactivating an endogenous cell gene which is located at the point of insertion of the foreign gene.
Depending on the function of the product of the inactivated gene, such an inactivation may lead to extensive morphological or physiological disorders in the transgenic animal, or may even prevent its survival.
On the other hand, the inactivation of a gene might be considered to be advantageous if the gene in question codes for a receptor of a virus or other infectious agent.
The inventors have studied the possibility of avoiding the disadvantages described above and associated, in some cases, with the possible inactivation of one or several endogenous cell genes with an important function in the course of the production of transgenic animals.